Photoaffinity labeling is a method which has been successful for the identification and localization of macromolecular receptors. For the isolation of peptide receptors and the study of peptide-protein interactions, a chemically-stable amino acid photoaffinity probe which can be incorporated into peptide ligands by both solid phase and solution peptide syntheses is needed. Additional desirable properties include (1) activation by low energy light (300-350 nm), (2) rapid reaction of the activated intermediate, (3) stability in an aqueous environment. The reagents described herein meet these requirements.
The products of this invention are reagents which allow the m or p-benzoylphenylalanine (m or p-Bpa) moiety to be incorporated into a peptide sequence by conventional solid or solution phase peptide synthetic methods. The resulting photoreactive peptide can be used to probe biological recognition sites (e.g. drug receptors or enzyme-active sites) by activation with low energy (300-350 nm) ultraviolet light. The benzoylphenylalanine reagents of this invention will probably find their chief application as laboratory tools. As such, these reagents are useful as photoaffinity labels for studies of the interaction of peptides such as neuropeptide analogs with proteins such as receptors, or they can be used to purify or separate enzymes or receptors from the total population of such materials.
The reagents of this invention are superior to conventional photoaffinity-labeling reagents containing the azide, diazo, or diazirine groups because these conventional reagents suffer from one or more of the following: They are chemically unstable, and inconvenient for solid phase synthesis. They are unstable to normal room lighting conditions or require high-energy light for activation which may result in damage to adjacent biological structures. They react by a one-step process involving the loss of a nitrogen atom and once activated cannot be reactivated. They may rearrange to form a slow-reacting intermediate which may permit the activated peptide to diffuse away from the biological recognition site before establishing a covalent linkage which results in non-specific labeling. On photoactivation, they may form a reactive species which reacts preferentially with water rather than on an adjoining biological structure.
The particular advantages of the benzoylphenylalanine reagents of this invention over previously available photoreactive amino acids are (1) their high chemical stability so that they can be used in conventional solid phase peptide synthesis; (2) their low photoreactivity with water; (3) their activation by low energy light; and (4) their stability in an aqueous environment.
p-Benzoylphenylalanine itself has been previously synthesized by R. E. Galardy [(1973) Ph.D. Thesis, Rockefeller University] but was isolated in very low yield and was prepared via a different synthetic method. The acylated derivative shown below was also disclosed in the thesis. These compounds were not incorporated into peptides because they could not be prepared in large enough quantities. ##STR3##
An advantage of the compounds of the present invention is the readily cleavable protecting group which can be removed leaving the rest of the peptide intact.